Biological and chemical amplification strategies are key to the design of successful molecular imaging agents. Several such strategies have been described including a) enzymatically activated prodrugs, b) covalent target binding, c) intracellular trapping, d) conformational changes upon target binding, e) pH induced fluorescence or magnetic changes f) increased avidity through multivalency, g) amplifying reporters, h) unnatural biorthogonal chemical reporters and i) pre-targeting. Some of these strategies have been extraordinarily robust but few possess intrinsic selectivity, are universally applicable for different classes of targets or are clinically translatable. One emerging chemical strategy for in vitro bioconjugation has been cycloaddition ("click chemistry"). Unfortunately, conventional reactions (e.g. between and azide and an alkyne) require elevated temperatures, a Cu(I) catalysts to be efficient or are simply too slow for in vivo use. We and others have discovered and tested a number of novel ring constrained reactants as more universal in vivo click reagents. In these reactions, a tetrazine replaces the azide functionality and readily reacts with constrained dienophile ligands. We have shown that the norbornene/ tetrazine click reaction can proceed orders of magnitude faster (in seconds as compared to hours-days with previous azide/alkyne reactions) and that reactions are very selective and specific. In preliminary data we have shown efficacy and compatibility of reactants with live cells. Importantly and extending this concept to transcyclooctenes, we have now shown that the technique works for intracellular targets as well as for extracellular targets. In parallel, proof-of-principle experiments shown that the technology allows in vivo clicking. The goal of this application is to further build on this cutting-edge technology and to develop generic amplifying in vivo click reactions for molecular imaging. In aim 1 we will perform more comprehensive cell based screens to identify lead compounds and conditions for dienophile/tetrazine " fast click reactions. In a second aim we will apply optimized compounds (antibody and/or small molecule conjugated dienophiles and tetrazine-fluorochromes) and conditions to biologically relevant in vivo models and ask a number of questions which may ultimately aid in the more widespread application of the technology: How efficient is in vivo click chemistry and what is the detection threshold in vivo? How does the strategy compare to current gold standards? Can the approach be used to measure EGFR target inhibition? Can the approach be used to quantitative drug distribution? Can the technology be used for multichannel imaging of several molecular targets? These experiments are a logical extension of our preliminary work and will likely result in broad, new imaging platforms. PUBLIC HEALTH RELEVANCE: The proposed research represents a new method for in vivo imaging of intra- and extracellular targets using biocompatible "click" reactions. The new method is very powerful as it harnesses very selective and specific chemistries and amplification strategies and has most recently been shown to work for intracellular targets as well.